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mouse gfap promoter sequence  (InvivoGen)
90
InvivoGen mouse gfap promoter sequence
Amplification of the putative Nkx2.1 binding sequences located in a 206 bp PCR product within the <t>GFAP</t> promoter (a) and in a 391 bp PCR fragment from the Lhx6 promoter (b) after chromatin immunoprecipitation (ChIP) with the Nkx2.1 antibody. Input DNA was added as the positive control as it contains the cross-linked sonicated genomic DNA taken before ChIP with the Nkx2.1 antibody; a strong signal was observed for all the promoter regions. No amplification of the Nkx2.1 <t>core</t> <t>sequence</t> (tcaag) located in two, 410 bp and 352 bp, PCR products from the Ngn2 promoter was detected (c) . No signal was detected in the negative control where no antibody was added for ChIP nor when no DNA was added while performing the PCR (a–c) . A very faint signal was detected in some samples immunoprecipitated with non-specific control IgG. These results suggest that Nkx2.1 binds in vivo to the promoter region of GFAP at putative Nkx2.1 binding sequences. The relative intensities of all the bands were calculated by assigning an arbitrary value of 1 to the input band. The quantifications are indicated below the gels. The Figure represents one of three independently performed assays. As a control, human embryonic kidney 293 (HEK293) cells were co-transfected with two reporter-constructs, namely, the pDRIVE-mGFAP-LacZ and the pCAG-IRES-Tomato plasmids (d–f) . Cell nuclei were counterstained in blue with Hoechst ( d ). In the control, only 4.26% of Tomato + cells were LacZ + , n = 94. To test the binding of Nkx2.1 to the GFAP promoter, HEK293 cells were co-transfected with two reporter constructs, namely, the pDRIVE-mGFAP-LacZ and the pCAG-Nkx2.1-IRES-Tomato plasmids (g–i) . Cell nuclei were counterstained in blue with Hoechst ( g ). Activation of the LacZ reporter was seen upon addition of the Nkx2.1 expression vector, thus confirming that Nkx2.1 activates the GFAP promoter, most probably by binding the sequence identified as Nkx2.1 consensus. Here, 98% of Tomato + cells were LacZ + , n = 50. Bar = 50 μm.
Mouse Gfap Promoter Sequence, supplied by InvivoGen, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Amplification of the putative Nkx2.1 binding sequences located in a 206 bp PCR product within the GFAP promoter (a) and in a 391 bp PCR fragment from the Lhx6 promoter (b) after chromatin immunoprecipitation (ChIP) with the Nkx2.1 antibody. Input DNA was added as the positive control as it contains the cross-linked sonicated genomic DNA taken before ChIP with the Nkx2.1 antibody; a strong signal was observed for all the promoter regions. No amplification of the Nkx2.1 core sequence (tcaag) located in two, 410 bp and 352 bp, PCR products from the Ngn2 promoter was detected (c) . No signal was detected in the negative control where no antibody was added for ChIP nor when no DNA was added while performing the PCR (a–c) . A very faint signal was detected in some samples immunoprecipitated with non-specific control IgG. These results suggest that Nkx2.1 binds in vivo to the promoter region of GFAP at putative Nkx2.1 binding sequences. The relative intensities of all the bands were calculated by assigning an arbitrary value of 1 to the input band. The quantifications are indicated below the gels. The Figure represents one of three independently performed assays. As a control, human embryonic kidney 293 (HEK293) cells were co-transfected with two reporter-constructs, namely, the pDRIVE-mGFAP-LacZ and the pCAG-IRES-Tomato plasmids (d–f) . Cell nuclei were counterstained in blue with Hoechst ( d ). In the control, only 4.26% of Tomato + cells were LacZ + , n = 94. To test the binding of Nkx2.1 to the GFAP promoter, HEK293 cells were co-transfected with two reporter constructs, namely, the pDRIVE-mGFAP-LacZ and the pCAG-Nkx2.1-IRES-Tomato plasmids (g–i) . Cell nuclei were counterstained in blue with Hoechst ( g ). Activation of the LacZ reporter was seen upon addition of the Nkx2.1 expression vector, thus confirming that Nkx2.1 activates the GFAP promoter, most probably by binding the sequence identified as Nkx2.1 consensus. Here, 98% of Tomato + cells were LacZ + , n = 50. Bar = 50 μm.

Journal: Scientific Reports

Article Title: Nkx2.1 regulates the generation of telencephalic astrocytes during embryonic development

doi: 10.1038/srep43093

Figure Lengend Snippet: Amplification of the putative Nkx2.1 binding sequences located in a 206 bp PCR product within the GFAP promoter (a) and in a 391 bp PCR fragment from the Lhx6 promoter (b) after chromatin immunoprecipitation (ChIP) with the Nkx2.1 antibody. Input DNA was added as the positive control as it contains the cross-linked sonicated genomic DNA taken before ChIP with the Nkx2.1 antibody; a strong signal was observed for all the promoter regions. No amplification of the Nkx2.1 core sequence (tcaag) located in two, 410 bp and 352 bp, PCR products from the Ngn2 promoter was detected (c) . No signal was detected in the negative control where no antibody was added for ChIP nor when no DNA was added while performing the PCR (a–c) . A very faint signal was detected in some samples immunoprecipitated with non-specific control IgG. These results suggest that Nkx2.1 binds in vivo to the promoter region of GFAP at putative Nkx2.1 binding sequences. The relative intensities of all the bands were calculated by assigning an arbitrary value of 1 to the input band. The quantifications are indicated below the gels. The Figure represents one of three independently performed assays. As a control, human embryonic kidney 293 (HEK293) cells were co-transfected with two reporter-constructs, namely, the pDRIVE-mGFAP-LacZ and the pCAG-IRES-Tomato plasmids (d–f) . Cell nuclei were counterstained in blue with Hoechst ( d ). In the control, only 4.26% of Tomato + cells were LacZ + , n = 94. To test the binding of Nkx2.1 to the GFAP promoter, HEK293 cells were co-transfected with two reporter constructs, namely, the pDRIVE-mGFAP-LacZ and the pCAG-Nkx2.1-IRES-Tomato plasmids (g–i) . Cell nuclei were counterstained in blue with Hoechst ( g ). Activation of the LacZ reporter was seen upon addition of the Nkx2.1 expression vector, thus confirming that Nkx2.1 activates the GFAP promoter, most probably by binding the sequence identified as Nkx2.1 consensus. Here, 98% of Tomato + cells were LacZ + , n = 50. Bar = 50 μm.

Article Snippet: The mouse GFAP promoter sequence from the pDRIVE-mGFAP plasmid (#pdrive-mgfap, InvivoGen) used for transfection experiments is shown in .

Techniques: Amplification, Binding Assay, Chromatin Immunoprecipitation, Positive Control, Sonication, Sequencing, Negative Control, Immunoprecipitation, In Vivo, Transfection, Construct, Activation Assay, Expressing, Plasmid Preparation